Stimulation of CD28 alone has been shown to regulate cytokine gene transcription and expression of the type 1 insulin-like growth factor receptor (IGF-1R) in lymphocytes. In this study, the ephrin receptor tyrosine kinase ephA3, was identified as a new CD28-responsive gene in Jurkat cells by using a human cytokine/receptor array. EphA3 was not detected in normal peripheral T cells, in any subset of thymus-derived developing T cells, or in Hodgkin's lymphoma. However, contrary to previous findings, EphA3 was detected in a panel of T-cell lymphomas. Stimulation of Jurkat cells with ephrin-A5 resulted in loss of cell adhesion to fibronectin and recruitment of the adapter protein CrkII to EphA3. Interestingly, EphA3 expression in CD28-stimulated Jurkat cells was enhanced by IGF-1 or by overexpression of the IGF-1R, and was suppressed by anti-IGF-1R blocking antibodies. The data suggest that CD28- and IGF-1-regulated expression of EphA3 is associated with adherence and that it may be involved in the motility of malignant T cells.